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October 2000

Indirect and Direct Antiglobulin (Coombs) Testing and the Crossmatch

Darrell J. Triulzi, M.D.
Medical Director

                                                                                                           

INTRODUCTION

The antiglobulin test was first introduced into clinical medicine in 1945 by R.R. Coombs who showed that it could be used to detect non-agglutinating red cell antibodies (indirect antiglobulin test, IAT) or sensitized red cells (direct antiglobulin test, DAT).  Most non-agglutinating (incomplete) antibodies are IgG, although some antibodies are IgM. These antibodies do not spontaneously cause agglutination due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity.  The antiglobulin reagent is able to bridge these negative forces.  Current antiglobulin reagent (Coombs reagent) preparations contain a "cocktail" of monoclonal antibodies directed against human IgG and C3.  The latter is more effective than an anti-IgM reagent for detecting IgM antibodies because a single IgM molecule will bind numerous complement molecules to the red cell surface and IgM antibodies tend to spontaneously dissociate from the red cell membrane.

Direct Antiglobulin Test (DAT)

The DAT is used to detect IgG or C3 bound to the surface of the red cell.  In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.  Non-immune causes of hemolysis such as DIC, thrombotic thrombocytopenic purpura, mechanical hemolysis such as those due to artificial valves or burns, hemoglobinopathies (sickle cell, thalassemia), red cell enzyme deficiencies (G6PDP, pyruvate kinase), and red cell membrane defects (hereditary spherocytosis, PNH) will have a negative DAT. Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT. A positive DAT can occur without hemolysis. A small proportion of normal  individuals have a positive DAT without evidence of decreased red cell survival.  Thus, a positive DAT, by itself, does not mean that the patient has an immune hemolytic anemia. 

The DAT is a 5-10 minute procedure performed by incubating patient red cells with the antiglobulin (Coombs) reagent.  A positive DAT due to IgG is seen most frequently in patients with warm autoantibodies.  Approximately 1/3 of these patients also have C3 on the red cell membrane.  IgG coated red cells may also be seen in patients who have received an incompatible transfusion.  Thus, the DAT is routinely performed as part of all transfusion reaction investigations.  IgG bound to the red cell surface can be eluted and its specificity determined.  Eluted autoantibodies usually bind to all red cells (panagglutinin) but occasionally have specificity within the Rh system.  Eluted alloantibodies can usually be given a distinct specificity.(eg. anti-D, anti-K)  Red cells sensitized with IgG may be destroyed by extravascular hemolysis.  The primary site of removal is the spleen via Fc receptors on phagocytic cells.  Factors that determine whether hemolysis will occur include: antibody titer, number of IgG molecules on the red cell, number of antigen sites on the red cell, IgG subclass, and splenic function.   

A positive DAT due to complement (C3) alone is seen in patients with cold autoantibodies, paroxysmal cold hemoglobinuria, and in some drug induced hemolytic anemias.  The offending antibodies are typically of the IgM isotypes that efficiently bind complement.  IgM antibodies are not directly detected by the DAT, but are detected indirectly by the presence of C3 on the red cell surface.  Cold autoimmune hemolytic anemias may be associated with intravascular hemolysis due to complement mediated lysis.  Extravascular removal of C3 coated cells can also occur via complement receptors on phagocytes in the liver. 

Rarely, patients with immune hemolysis may have a negative DAT.  Many of these patients have IgG, IgM, or IgA antibodies detectable on the red cell only with more sensitive techniques. (MicroCoomb’s test)

Indirect Antiglobulin Test (IAT)

The IAT is used to detect red cell antibodies in patient serum.  In clinical practice this is referred to as the "antibody screen" and is part of the type and screen procedure.  Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.  Most clinically significant alloantibodies are IgG antibodies that react best at 37°C and are formed as a result of previous exposure via transfusion or pregnancy.  Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell antigens.  IgM antibodies are usually not clinically significant (except for ABO antibodies) but are a source of in-vitro serologic difficulty that may delay transfusion.  Examples include antibodies to the Lewis, I, P, M, and N red cell antigens.  IgM antibodies react best at cold temperatures (4°C) and are usually naturally occurring in that they do not require a sensitizing event.   

The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination.  If the antibody screen is positive, additional testing is required to determine the specificity of the antibody. 

If transfusion is necessary, patients with clinically significant red cell alloantibodies identified in the antibody screen should receive antigen negative red cells.  Compatible blood may be difficult to find if antigen negative blood is rare or if multiple antibodies are present.  Consultation with the transfusion service is helpful in developing a transfusion strategy in these cases.

CROSSMATCH

The crossmatch (CM) represents a special form of the IAT in which the red cells used for testing are from the unit intended for transfusion. The purpose is to establish in vitro compatibility in the expectation that the transfused cells will exhibit normal in-vivo survival.  Historically, a major CM involving patient serum and donor red cells was performed on every unit intended for transfusion. The major crossmatch takes 20-30 minutes and is now reserved only for patients with clinically significant red cell antibodies. The minor CM involving patient red cells and donor plasma has not been performed for more than 20 years because packed red cells have <70 ml of plasma. More recently it has been recognized that patients with a negative antibody screen and no history of red cell antibodies do not require a complete 20-30 minute crossmatch.  The chances of  a clinically significant red cell antibody being missed in a patient with a negative antibody screen (false negative) is 1-4/10,000. Approximately 95% of transfusions occur in patients with a negative antibody screen.  Such patients can undergo abbreviated CM testing in which only ABO compatibility of the unit need be established.  There are two methods for abbreviated CM testing.  The “immediate spin” crossmatch (ISCM) requires only a 5 minute incubation at room temperature with patient serum and donor red cells. For the last 4 years the American Association of Blood Banks has also allowed an abbreviated CM to be performed by computer confirmation of ABO compatibility without any serologic testing: ”computer crossmatch”.  The patient must have two determinations of their ABO group on record and a negative antibody screen. Using a validated computer system the bar code on unit of blood can be wanded and the computer will compare the ABO of the unit to that of the patient and indicate whether it is compatible.  Advantages of the computer crossmatch includes: faster turn-around, computer prevents release of ABO incompatible units, lower reagent costs, and improved quality control.

 

Summary

The IAT and DAT are used to detect red cell antibodies in the serum and on the red cell, respectively.  The DAT is used to determine whether patients with hemolysis have an immune etiology.  The IAT is used to identify clinically significant red cell alloantibodies that are important in choosing compatible blood products.

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Copyright © 2000, Institute For Transfusion Medicine



For questions or further information on regarding
Indirect and Direct Antiglobulin Testing, please contact Darrell J. Triulzi, M.D. at The Institute For Transfusion Medicine: 412-209-7320. 

Copies of previous Transfusion Medicine Update issues can be obtained from our web page: www.itxm.orgTo be placed on our mailing list for a hard copy, please contact Deborah Small by e-mail: dsmall@itxm.org or by phone: (412) 209-7320.